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Chapter 18

DNA PROBES

J. Castelino

Introduction

There is no doubt that radioimmunoassays have contributed a lot to the diagnosis and
understanding of many diseases. There are two problems with them:

(a) they use radioisotopic label and

(b) they are difficult to automate because of the separation

step, which is indispensable.

To bring the immunoassays in the fold of clinical chemistry, a large amount of effort is
now directed towards developing non-isotopic assays.

On the other hand, In vitro nuclear techniques are gradually moving in new directions
opened up by modern molecular biology. Two approaches are promising in this respect:

(a) DNA probes and

(b) proteins of biological interest produced by

recombinant techniques.

The creation of DNA probes for detection of specific nucleotide segments differs from
ligand detection in that it is a chemical rather than an immunological reaction.
Complementery DNA or RNA is used in place of the antibody and is labelled with 32P. So
far, DNA probes have been successfully employed in the diagnosis of inherited disorders,
infectious diseases, and for identification of human oncogenes.

Recombinant techniques have already provided a number of pure human proteins in large
quantities including insulin, growth hormone, lymphokines etc. Immunoassays, mostly RIA,
have been critical in monitoring therapeutic blood transfusion.

The latest approach to the diagnosis of communicable and parasitic infections is based
on the use of deoxyribonucleic acid (DNA) probes. The genetic information of all cells is
encoded by DNA and the DNA probe approach to identification of pathogens is unique
because the focus of the method is the nucleic acid content of the organism rather than the
products that the nucleic acid encodes. Since every properly classified species has some
unique nucleotide sequences that distinguish it from every other species, each organism's
genetic composition is in essence a finger print that can be used for its identification. In
addition to this specificity, DNA probes offer other advantages in that pathogens may be
identified directly in clinical specimens; the method does not depend on the expression of an
antigen, and organisms that may have undergone spontaneous mutation can still be identified

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since single mutations rarely result in a major change in nucleic acid composition. In order
to monitor the end result of the DNA probe test, a label is incorporated into the probe. The
most common label is the radionuclide "Phosphorus, that is detected in the end product of
the test either by autoradiography or with a scintillation counter.

Clinical problems both diagnostic and therapeutic are being solved using new approaches
made possible by DNA techniques. These techniques are over a decade old, yet many
clinicians and biomedical personnel are unaware of the potential impact of these techniques
on their ability to diagnose diseases rapidly and accurately. During the next decade, these
techniques are expected to become the backbone of diagnostic laboratories. Preliminary
reports from laboratories already using commercially available DNA probe kits are
encouraging. This Chapter tries to introduce these technology to the physicians, describe
their potential for medical diagnosis and point out how relevant and practicable they are for
the developing countries.

The DNA Molecule

The basis of molecular biology is deoxyribonucleic acid or DNA. DNA is a

double-stranded helical molecule composed of pairs of nucleotide bases: adenine (A) guanine
(G) thymine (T) and cytosine (C). Within a strand of DNA, the bases are linked by a
sugar-phosphate backbone. The DNA molecule resembles a twisted ladder with the A and
T and G and C linked together by hydrogen bonds. The two strands can be separated by heat
(thermal denaturation) or by raising the pH or lowering the ionic strength of the DNA
solution. The DNA molecule is most stable in its native double-stranded state. Thus when
single-stranded DNA is placed in solution, under appropriate temperature and salt conditions,
the complementary strands will recombine to form a duplex molecule (Fig. 18.1)

A region of DNA, which encodes a protein is termed a gene. The genetic information
is encoded by a sequence of bases via a non-overlapping code in which three bases (a triplet)
determine a particular amino acid. For a gene to be expressed, an enzyme, RNA polymerase
II, copies or transcribes one strand of the DNA into mRNA (messanger RNA), which is then
decoded or translated by the protein synthesis machinery in the cytoplasm. The mRNA
comprises of a single-stranded poly nucleotide chain with a sugar-phosphate backbone in which
the order of bases is the complement of the transcribed DNA strand of the gene. In RNA,
thymine (T) is replaced by a closely related base uracil (U) which will also pair with or
hybridize to Adenine (A).

DNA probes, then, are pieces of nucleic acid, labeled in some fashion, that can seek out
and bind to stretches of DNA or RNA that have complementary sequences (adenine opposite
thymine (or uracil), cytosine opposite guanine. The two strands of nucleic acid must be in
contact and have sufficient complementary base sequences so that a stable double-stranded
molecule is formed. Complementary sequences of DNA can bind to RNA counterparts.
Thus probes may also be labeled RNA strands directed towards DNA targets or labeled DNA
sequences directed towards RNA targets.

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 DNA PROBES

Nucleic acid molecules will recombine only when the two strands are composed of
complementary sequences. It is this property of nucleic acids that is the basis of the DNA
or RNA probe reaction. The site of action is shown in Fig. 18.2.

The hybridization reaction consists of four components: the probe, the target (which is
contained in the sample), the reporter molecule, and the hybridization method. The sample
serves as a source of nucleic acid to be analysed and can consist of a suspension of an
unknown organism, (for culture confirmation) or a clinical specimen such as sputum, or
serum. The nucleic acid in the sample is referred to as the target DNA or RNA, and the
radionuclide label on the probe, the reporter molecule.

Designing a Probe

All organisms contain some unique sequences of DNA or RNA within their genome that
distinguish them from all other organisms. The key to developing a nucleic acid probe (i.e.
either a DNA or a RNA probe) is to isolate these sequences, reproduce them in large
quantities, and attach a reporter molecule to them so they can be incorporated into a
hybridization reaction. Hybridization is a process whereby two single strands of nucleic acid
come together to form a stable double-stranded molecule. As long as the sequences of bases
along each stretch of nucleic acid are complementary, they will bind and stay together.

To produce the unique sequences, cloning vectors are used. The most commonly used
cloning vectors are Plasmids. These plasmids are co-valently closed circular pieces of DNA
that replicate independently of the bacterial chromosome. They are not required for cell
replication but often give the host cell some advantage such as antimicrobial resistance, etc.
Plasmids range in size from a few thousand base pairs (bp) to as large as 400 000 bp
(400 kilobases or kb). One set of plasmids often used in recombinant DNA technology is
called cloning vectors. Cloning vectors are small plasmids often just two to five kb that
contain a selectable marker such as ampicillin resistance and a stretch of DNA that can be
cleaved by many different restriction endonucleases. These are enzymes, found in bacteria,
which cut DNA at specific sequences. For example, the enzyme EcoRI cuts the DNA chain
between G and A in the sequence GAATTC. Each time a particular DNA is cleaved by an
enzyme, precisely the same set of fragments is generated. Many such enzymes are now
available.

Examples: p6r322, a 4.6 kb plasmid with ampicillin and tetracycline

resistance; pUC18 and pU19 a pair of plasmids with
ampicillin resistance, and multiple restriction sites and an
indicator system utilizing a 6 galactisidase gene where
plasmids with foreign DNA inserts produce colourless
colonies, while plasmids with no inserts produce blue
colonies (Figs. 18.3 and 18.4).

The usual method of isolating and reproducing the unique sequence that will become the
probe begins by cleaving that stretch of nucleotide bases away from the remaining nucleic

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acid in the cell, using a set of enzymes known as restriction endonucleases. These enzymes
provide the molecular scissors to cleave DNA at specific sequences of nucleotide bases. For
instance, the enzyme Bam HI, obtained from Bacillus amyloliquefaciens, will cleave the
DNA molecule between the Guanine - Guanine bases, Hae III, from Haemophilus aegyptius,
between guanine and cytosine, and the ECORI, from Escherichia coli, between Guanine and
adenine. Endonucleases are named by the first letter of the genus, the first 2 letters of the
species, the strain and the number indicates whether it is the first, second etc. enzyme
discovered in the organism. The single-stranded segments produced by restriction
endonuclease cleavage are referred to as sticky ends because they can recombine with any
other piece of DNA that has been cleaved with the same enzyme, regardless of the source of
that DNA. Thus DNA from a virus such as herpes simplex (HSV) type 2 can be cleaved and
inserted into a small plasmid from E. coli if the plasmid has been cleaved with the same
enzyme. The sticky ends are then sealed with a second enzyme known as DNA ligase, to
produce a double-stranded circular molecule and introduced into E. coli by transformation
(addition of CaCl2). The plasmid containing the DNA insert of HSV type 2 will replicate in
E. coli making hundreds of copies. This process is referred to as cloning. In the simplest
terms, cloning is the process of isolating a piece of DNA and placing it in a vector that
allows hundreds of copies of that DNA sequence to be produced, when the plasmid replicates
(Figs. 18.5 and 18.6).

The plasmid now greatly amplified in copy number can be purified from the bacterial cell
by centrifugation or filtration or by column filtration. The plasmid can be labeled directly
by random primer method or the foreign DNA sequence isolated by restriction endonuclease
digestion followed by centrifugation and the probe labelled by nick translation.

The unique sequence that will constitute a diagnostic probe need not be a whole gene nor
need it be from within a sequence that actually encodes a protein.

Hybridization Reactions

After a double-stranded DNA molecule is denatured to single strands, it is capable of

reassociating with either a DNA or a RNA strand of complementary sequence. The degree
and specificity of binding depends on temperature, pH, use of a denaturant such as
formamide, and salt concentration of the reaction buffer. Nucleic acid molecules can tolerate
a certain number of mismatched base pairs (such as adenine molecules lining up opposite
cytosine or guanine molecules opposing thymines) and still form stable duplexes as long as
a significant number of base pairs do match and form bonds. However, the greater the
degree of mismatched bases along the strands of nucleic acid, more likely that the two
molecules would come apart. The degree of mismatch that can be tolerated in a hybridization
reaction and still maintain a double-stranded molecule (and produce a positive hybridization
signal) is referred to as the "stringency" of hybridization.

The concept of stringency is very important in understanding the specificity of DNA

probe reactions. If the salt concentration or temperature of hybridization is altered, the
specificity of the probe will change. The range of conditions that can be tolerated without

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 DNA PROBES

affecting the specificity of a probe vary depending on the length of the probe and the
percentage of guanine and cytosine residues in the probe. The shorter the probe, the more
narrow the range of temperature and salt concentration that can be tolerated.

Under conditions of high stringency, only exact matches of DNA will anneal and stay
together. Under conditions of low stringency (i.e. reactions carried out at low temperature,
in high salt concentration, or in low concentration of formamide) two DNA strands that are
only 80 to 90% homologous may bind together and result in a positive hybridization signal.
For example, under conditions of high stringency, a probe developed to Campylobacter jejuni
would hybridize only to DNA from that organism while under conditions of low stringency
the same probe would bind to DNA from Campylobacter coli and C. fetus but still not bind
to DNA from E. coli or Shigella flexneri.

Formats for Hybridization Reactions.

Hybridization reactions can be performed in four formats: on a solid support (filter), in

solution, in situ, or by using the Southern Blot hybridization procedure after gel
electrophoresis. The majority of DNA probes reported in the literature have used the solid
support format. However, both solution and solid support formats are utilized by the new
commercially available probe kits.

To carry out the hybridization using the solid support format, the sample, which can be
purified DNA, a suspension of a microorganism isolated in culture, or a clinical specimen,
is either spotted directly on the filter or placed in a vacuum manifold which concentrates the
specimen in a small area. The sample is lysed and the DNA denatured by the addition of
NaOH or by steaming the filter above a beaker of ammonium acetate. Acid-fast organisms
may require pretreatment with anti-microbial agents or other reagents to aid in disrupting
their cell wells. Once denatured, the DNA is attached to the filter by baking if in a vacuum
oven at 80°C for two hours. The filter is prehybridized with a non-homologous DNA such
as salmon sperm DNA or calf thymus DNA, to prevent the non-specific binding of probe to
the filter. After hybridization, the filters are washed at various temperatures determined by
the stringency of the reaction. Although nitrocellulose filters are traditionally used for such
assays, Whatman No. 541 paper and synthetic nylon filters are also frequently used
(Figs. 18.7 and 18.8).

The second format is to carry out the hybridization reaction in solution. In this format,
both the target and the probe nucleic acid are free to move, maximizing the chance that
complementary sequences will align and bind. Solution hybridizations go to completion 5 to
ten fold faster than on solid supports. In several of the commercially available solution
hybridization reactions samples are incubated at high temperature (72 °C) in a sonicating water
bath to disrupt the cells and cause them to release their nucleic acids. The addition of glass
beads to the sample often aids in the disruption of the cell wall. After the hybridization step,
the nascent duplexes are removed from solution by the addition of hydroxyapatite, which
selectively binds duplex nucleic acid leaving single-stranded nucleic acids in solution. In
some procedures the duplex DNA is removed from the hydroxyapatite by increasing the salt

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concentration or by heating, while in other procedures the hydroxyapatite-bound

doulbe-stranded nucleic acid is collected by centrifugation and the pellet is washed and
quantitated by scintillation counting.

Polymerase chain reaction (PCR)

At times the amount of target nucleic acid present in a sample may be extremely small
and thus beyond the limits of detection for conventional hybridization reactions. The recent
development of the polymerase chain reaction is one of the most substantial technical
advances in molecular genetics in the past decade. The procedure amplifies DNA by
chemical proliferation in vitro of a predetermined stretch of DNA. It is possible to amplify
specific DNA sequences, from as short as 50 base pairs to over 2000 base pairs in length,
more than a million fold in only a few hours.

The PCR method was developed by Saikai and his co-workers at Cetus Corporation. It
is based on the repetitive cycling of three simple reactions, the conditions of which vary only
in the temperature of incubation. All three reactions occur in the same small tube and with
temperature stable reagents. The repetitive cycle is therefore self contained and fully
automated.

In addition to the target DNA to be amplified, the important reagents are two single
stranded oligonucleotide (Primers), synthesized to be complementary to known sequences of
the target DNA; larger amounts of the four deoxyribonucleoside triphosphates, and the heat
stable Taq DNA polymerase, isolated from the thermophilic bacterium Thermus aquaticus.

The first step in the procedure is the heat denaturation of native (target) double-stranded
DNA, which can be used virtually straight from any clinical, laboratory, or forensic
specimen. The target DNA melts at high temperature (90 - 100°C) to liberate single-stranded
DNA, which can subsequently reanneal to any other DNA that has complementary sequences.
Recent experience suggests that amplification may begin in a sample containing only a single
target molecule of DNA, making the PCR the most sensitive detection technique for specific
DNA sequences.

In the second step of the cycle, performed at reduced temperatures, two short DNA
primers are annealed to complementary sequences on opposite strands of the target DNA.
These primers are chosen to encompass the desired genetic material; they define the two ends
of the amplified stretch of DNA. The two primers must not anneal to each other and their
sites of annealing must be sufficiently distant from each other to allow the subsequent
synthesis of new products. The specificity of the PCR method is derived from the precision
of this DNA-DNA annealing reaction.

The cycle's third step is the actual synthesis of a complementary second strand of new
DNA, which occurs through the extension of each annealed primer by Taq polymerase in the
presence of excess deoxyribonucleoside triphosphates. A new single strand of DNA is
synthesized for each annealed primer. Each new strand consists of the primer at its 5' end

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 DNA PROBES

trailed by a string of linked nucleotides that are complementary to those of the corresponding
template. An essential feature of the PCR procedure is that all previously synthesized
products act as templates for new primer extension reaction (i.e. DNA synthesis) in each
ensuing cycle. The result, aptly named "chain reaction" is the geometric amplification of new
DNA products (Fig. 18.9).

Since the primers form the kernals of all new DNA strands, each of the two different
primers, as well as the four deoxyribonucleoside triphosphates must initially be present in
massive amounts relative to the quantity of target DNA.

After extension of the primers, the cycle is repeated first by raising the temperature to
convert double-stranded to single-stranded DNA, and then by lowering the temperature to
allow the steps of annealing and extension.

The success of the procedure depends on knowing the sequence of the target DNA, at
least at the site of primer annealing to allow the synthesis of appropriate complementary
primers. Synthesizing the olignucleotide primers is itself an automated procedure and
relatively inexpensive. If a RNA sequence is to be amplified a cDNA copy of it must be
synthesized first using reverse transcriptase, before the PCR is begun.

A PCR reaction cycle typically takes five to seven minutes and is repeated 30 to 40 time
to give a million copies.

Oligonucleotide Probes

Some of the currently available probe kits utilize very small stretches of nucleotides as
probes. These short probes (oligonucleotide probes) are synthesized de novo in the laboratory
on one of the several instruments designed for this purpose. Normally, only 14 to 40 base
pair in length, these probes display exquisite specificity. Under stringent conditions they may
be capable of detecting a change in a single base pair of a DNA or RNA sequence, which is
enough to prevent binding of the probe to the targets.

Olinucleotide probes are very stable over time, and relatively simple to prepare. One
batch of oligonucleotide probes is often enough to last in a laboratory for several years since
only picogram amounts of probe are required for hybridization. Because of their small size
and low complexity (base ratios) these short probes hybridize to target DNA at very rapid
rates often with reaction times of less than 30 mins. This is in contrast to long probes that
often require 4 to 16 hours.

The disadvantage of short probes is that each oligonucleotide can be labelled with only
a single reporter molecule. Thus they are often 10 to 100 fold less sensitive than long
probes.

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 CHAPTER 18

Labelling Probes

Traditionally the most commonly used detection system is the 32P label directly
incorporated into the probe by nick translation. Other methods and other labels may also be
used (Table I).

TABLE I: COMMON RADIOLABELS FOR DNA PROBES

Isotope Emission Energy T'/2 dpm/mol

Type (100% isotopic enrichment)

32p B 1.71 MeV 14.3 d 2.02 x 1019

125
I 7 0.035 MeV 60 d 3.94 x 10"
3
H B 0.0181 MeV 12.3 d 6.39 x 1016
35
S B 0.167 MeV 87.4 d 3.33 x 101*

Nick translation

Incorporation of labelled bases into long probes by nick translation involves nicking one
of the two strands of double-stranded DNA with DNASE 1, and then excising stretches of
the single strands at the nicks with the 5'-3' exonuclease activity of E. coli DNA
polymerase I. In the process the polymerase enzyme also adds nucleotides to the 3' OH.
The native bases are replaced with labelled bases in the reaction mixture as the nick moves
along the DNA strand. When 32P-labelled bases are incorporated, specific activities of 5 x
108 dpm/fxg can be obtained with labelled strands 400 to 800 nucleotides in length. Probes
synthesized with 3H labelled bases have specific activities of 0.5 x 108 to 1.5 x~108 dpm//ag.
Labelling by nick translation is a fairly simple reaction resulting in uniformally labelled
probes with high specific activity (Fig. 18.10).

Random Primer Method.

This method is based on the hybridization of the DNA to be labelled with a mixture of
all the possible hexanucleotides. The complementary strand is synthesized from the 3' end
of the primer hexanucleotide by the Klenow fragment of E. coli DNA polymerase I. The
unique sequence need not be separated from the plasmid. The plasmid DNA containing the
unique sequence to be used as a probe is heated to separate into single-stranded DNA. The
solution is then placed on ice and two to five /xl of random primers are added. These primers
will anneal to portions of the two single-stranded circular DNAs. Then 5 /xl of 32P dATP and
unlabelled dCTP, dGTP and dTTP are added with DNA polymerase I. The primers thus
extend and a new strand of DNA is produced incorporating the 32P label. The unbound 32P
dATP is removed by passing through a spin column (a small sephadex bead column placed
on top of a centrifuge tube and spun). The DNA comes through the 32P dATP and other
triphosphates remain on the column.

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 DNA PROBES

The labelled plasmid DNA is boiled shortly before the hybridization reaction and a drop
placed on the spotted target DNA on nitrocellulose paper.

Detection Systems

Autoradiography

The extent of hybridization with radiolabelled probes on filters and in situ is monitored
with autoradiographic methods. The procedure is sensitive with good resolution and does not
destroy the hybridization support matrix. It lacks the exact quantitative ability of liquid
scintillation methods, but it can reveal artifacts not seen in counting.

The hybridized filter is exposed to X-ray film (Kodak XAR 5 or X-OMAT AR) in a light
tight cassette. Exposures range from hours to days, depending on the level of radioactivity
present. In general, an area of a dot 3 mm across with about 100 dpm of 32 P. The sensitivity
of the method can be increased with the use of intensifying screens (Du Point Cronex Quanta
III, Cronex Lightning Plus, or Fugi Mach II) by eight to ten fold with two screens.

The film, placed between the hybridized filter and screens, is exposed at -70°C in order
to prolong the fluoresence emitted in response to a decay event. As little as 5dpm/7mm2 area
can be visualized overnight.

Scintillation Counting

If the hybridized support is sufficiently radioactive, the area of interest can be cut out and
counted in a liquid scintillation counter.

Probes in a Diagnostic Laboratory

Current DNA probes require one to three hours to complete. This is still considered an
advantage when compared to culture methods that may take four to six weeks. Probes enable
the direct detection of pathogens in clinical specimens although often not the sensitivity of the
pathogen to drugs. In certain cases, e.g. P. falciparum and S. mansoni, Neisseria
gonorrhoeae sensitivity to pyrimethamine, oxaminiquine and pencillin can be determined by
using probes designed for this.

Tests using probes when used in a batch format are relatively inexpensive, although use
of probes for individual specimens can be expensive (Table II).

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 CHAPTER 18

TABLE II. COMPARISON OF COSTS FOR 5 AND 50 SAMPLES IN THE

FILTER HYBRIDIZATION ASSAY FOR CAMPYLOBACTER JEJUNI

Cost Factor Co st per

5 samples 50 samples

DNA probe kit ($ 16) 3.20 0.32

Reagents ($ 5) 1.00 0.10
Labour ($ 19.50) 3.90 0.39

Total 8.10 0.81

SUGGESTED READING

[1] STEELE, C M . , DNA in medicine, Lancet U (1984) 908-912.

[2] WORLD HEALTH ORGANIZATION, The use of DNA probes for malaria
diagnosis: memorandum of a WHO meeting, WHO Bulletin,
64(1986)641-652.

[3] TENOVER, F.C., Diagnostic deoxyribonucleic acid probes for infectious

diseases. Clin. Microbiol. Rev. 1:1 (1988) 82-101.

[4] HOLMBERG, M., SHENTON, F.C., FRANZEN, L., JANNEH, K.,

SNOW, R.W., PETTERSSON, U., WIGZELL, H., GREENWOOD, B.M.,
Use of a DNA hybridization assay for the detection of Plasmodium falciparum
in field trials, Am. J. Trap. Med. Hyg. 3.7:2 (1987) 230-234.

[5] WIRTH, D.F., ROGERS, W.O., BARKER, JR., R., DOURADO, H.,
SUESEBANG, L., ALBUQUERQUE, B., Leishmaniasis and malaria: new
tools for epidemiologic analysis, Science, 224 (1986) 975-979.

[6] SIM, B.L.K., MAK, J.W., CHEONG, W.H., Identification of Brugia malavi
in vectors with a species-specific DNA probe, Am. J. Trop. Med. Hyg.
35 (1986) 559-564.

[7] ROGERS, W.O., BURNHEIM, P.F., WIRTH, D.F., Detection of

Leishmania within sand flies by kinetoplast DNA hybridization,
Am. J. Trop. Med. Hyg. 39:5 (1988) 434-439.

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 DNA PROBES

[8] PANYIM, S., YASOTHORNSRIKUL, S., TUNGPRADUBKUL, S.,

BAIMAL, V., ROSENBERG, R., ANDRE, R.G., GREEN, C.A.,
Identification of isomorphic malaria vectors using a DNA probe,
Am. J. Trop. Med. Hyg. 3.8:1 (1988) 47-49.

[9] COLLINS, F.H., PETRARCA, V., MPOFU, S., BRANDLING-BENNETT,

A.D., WERE./J.B.O., RASMUSSEN, M.O., FINNERTY, V. "Comparison
of DNA probe and cytogenetic methods for identifying field collected
Anopheles gambiae complex mosquitoes". Am. J. Trop. Med. Hyg.
39:6 (1988) 545-550.

[10] WITHERSPOON L.R., Radioimmunoassayists must embrace new technology,

J. Nuc. Med., 30 (1989) 1571 -1573.

[11] INNIS,M.A.,GELFAND, D.G.,SNINSKY, J., WHITE, T., PCR Protocols,

Academic Press, ISBN 0-12-372181-4 (1990).

[12] EISENACH, K.D., CAVE, M.D., BATES, J.H., CRAWFORD, J.T.,

Polymerase chain reaction amplification of a repetetive DNA sequence specific
for Myocardium tuberculosis, J. Infect. Dis. 161 (1990) 977-981.

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 CHAPTER 18

DNA HYBRIDIZATION TECHNIQUE

A 8

Native DNA Denature DNA to Binding of labelled probe

single strands to target DNA

Fig. 18.1 Structure of deoxyribonucleic acid, effects of denaturation and binding of

probe to target DNA [from Tenover, F.C., Clin. Microbiol. Rev 1,
83 (1988)].

ene probes ^

Cyiopljim.

Antibodies

Fig. 18.2 Site of action of gene probes.

284
 DNA P R O B E S

Bam HI
-GGATCC - G GATCC-

-CCTAGG - CCTAG G-
t

Hae I I I
GGCC - GG cc-
CCGG V - CC GG-

Fig. 18.3 Sequences cleaved by the restriction enzymes Bam HI and Hae III.

The Restriction Endonuclease

EcoRI

plasm.d
ONA

ONA cleavfd.

ky ends
produced

j AATTCT
CTTAA G

Fig. 18-4 The restriction endonuclease Eco RI. [From Tenover, F.C., CHn.
Microbiol. Rev. 1, 83 (1988)].

285
 CHAPTER 18

asmidW) Cleavage ' 1

Tc resistance
Restriction
endonuclease

DNA ligase seals

necks

Transformation. Tc
selection

Recombinant
plasmid

Fig. 18.5 Use of DNA ligase to create a covalent DNA recombinant joined through
association of termini generated by Eco RI.

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 DNA PROBES

PROBE DEVELOPMENT AND ISOLATION

(iot*t* OH A
(o* cDNA}

TfSf storm piaif

into £ co<i

JL

Fig. 18.6 The development of probes from diverse infectious agents. [From
Tenover, F.C., Clin. Microbiol. Rev. 1, 83 (1988)].

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 CHAPTER 18

I. lake fln9er-«rlck 2. Use Infectea cells

olooo uncle from In taicrotltre olate.
Infected Individual.

o ooo o o

3. Blot sanole on nltro-

eellulose f1Iter * i m
dot-Clot aooaraius.

1. Incubate 10 n n . In
12 3 4 5 6 7
KaOH follo»w Oy 10
«ln. in buffer- botn
a< room teweralure.
DHA seoaratec into
sin«le-str3.ioeo OKA.
S. Bafce In own at SO "I
for I hour.

6. Add laoellec DK* prooe

';/ Kltrocf1lulose 11 li

for alnlaua of " lours

at 13 X to
labelled prooe to
target OKA.

ft* «< v Prooe hyorlOUed

to target D«A.

7. Kasn.

8. Cover M|tn auio-

racioQraonic elate

S. C>eveloo for 6 hours

1n Oirk room.

Pnoiocraoti of e^Dosea Diate

Fig. 18.7 Method used for filter hybridization of DNA probes to P. falciparum DNA.

288
 DNA PROBES

SOUTHERN BLOTTING

Agaros* GrI
El«rtrophorcsts

Paper towels
Cellulose

P SS DNA
X

Fig. 18.8 Technique of southern hybridization.

289
 CHAPTER 18

DMA

New
ONA

\±
Prime* extension
Native ONA Heal denatmaoon Primet anneakng

Fig. 18.9 First Round of the Polymerase Chain Reaction. The basic
polymerase-chain-reaction cycle consists of three steps performed in the
same closed container but at different temperatures. The elevated
temperature in the first step melts the double-stranded DNA into single
strands. As the temperature is lowered for the second step, the two
oppositely directed oligonucleotide primers anneal to complementary
sequences on the target DNA, which acts as a template. During the third
step, also performed at a lower temperature, the Taq- polymerase
enzymatically extends the primers covalently in the presence of excess
deoxyribonucleoside triphosphates, the building blocks of new DNA
synthesis. The native DNA target sequences, which will be massively
amplified as "short products" in the ensuing cycles, are boxed. The vector
of action of the DNA polymerase is denoted by the arrows projecting from
the newly synthesized DNA, indicated by the dark bars.

290
 DNA PROBES

1 cc« /

DNA nicked or broken with deoxyribonuclease

32
P-labelled nucleotides A CGT added
with DNA polymerase I

NICKS repaired with new nucleotides replacing damaged ones

Fig. 18.10 Nick translation to make a DNA fragment sequence highly radioactive.

291